Synthesis and antitumor activity of substituted succinamides using a potato disc tumor induction assay.

In view of potential biological activities of some succinic acid derivatives, we synthesized some novel N-[4-(4-morpholinosulfonyl)-phenyl]-succinamides (6a, c; 7a, c) and N-[4-(benzylaminosulfonyl) phenyl]-succinamides (6b, d; 7b, d) derivatives as antitumor agents. The antitumor activity of compounds was studied using the potato disk bioassay technique. Vincristine at 0.25 mg/ml was employed as positive control and caused -67.24% inhibitions. Compound 7b at 1 mg/ml caused -80.50% tumor inhibitions with highest activity among compounds tested.

Potato disc is a useful test for monitoring the inhibition of crown-gall tumors (6,7). Crown gall is a neoplastic disease of plants induced by specific strains of the Gramnegative bacterium Agrobacterium tumefaciens (8), first reported by Smith and Townsend (9) and Jensen (10). This malignancy, normally affecting dicotyledonous plants, is induced by inoculation of a wound site with A. tumefaciens followed by the transfer of a large plasmid from A. tumefaciens to the plant (11). The genetic information (T-DNA) of the large plasmid transforms normal plant cells into autonomous tumor cells (12). Once the tumor induction has taken place, the autonomous proliferation of the tumor cells becomes entirely independent of the bacteria (13). Galsky (14) showed that inhibition of crown gall tumor initiation on potato discs showed good agreement with compounds and plant extracts known to be active in the 3PS (in vivo, mouse leukemia) antitumor assay. In addition to the inhibition of tumor initiation also the inhibition of the growth of the tumors agrees well with 3PS activity (15).

ORIGINAL ARTICLE
Activity of the compounds 6a-d, 7a-d was based on the method described above.

MAterIAls ANd Methods chemical
All the chemicals and reagents were purchased from Merck and Aldrich. 4-acetamidobenzenesulfonyl chloride was synthesized from acetanilide and chlorosulfonic acid according to published procedure. The IR spectra were measured on a Unicam SP-1100 spectrophotometer, using samples prepared as KBr disks. The 1 H NMR spectra were recorded on a Brucker AC-80 spectrometer (Germany) operating at a working frequency of 80 MHz, using tetramethylsilane as the internal standard. The samples were dissolved in DMSO-d 6 or CD 3 OD.
General method for preparation of 4-amino N-substituted benzenesulfonamides (4a, b). To 4-acetamido-N-substituted benzenesulfonamides (3a, b, 0.08 mol) in water (100 ml) was added concentrated HCl (50 ml). The mixture was heated for 1 h. until a clear solution was obtained. The heating was continued for additional 10 min. The clear solution was cooled to room temperature and neutralized with adding sodium hydroxide (10%) followed by saturated solution of sodium bicarbonate. A precipitate was formed which was filtered and washed with cold water and dried to give 4-amino N-substituted benzenesulfonamides (4a, b).
Methyl 3-chlorocarbonylpropionate (5). Succinic anhydride (10g, 0.1 mol) was refluxed in methanol (40 ml) for 8h. The excess methanol was evaporated in vacuum. An oily residue, succinic acid monomethyl ester (13.12 g) was obtained. Thionyl chloride (15 ml) was added dropwiseto the residue during 15 min, while stirring. The mixture was then refluxed for 5 h. Excess thionyl chloride was evaporated in vacuum. The greenish yellow liquid, methyl 3-chlorocarbonylpropionate was used directly in next step without any further purification.
N-substituted succinamic esters (6a, b). Compound 5 (4 ml, 0.02 mol) was added dropwise to the stirring solution of compound 4 (0.02 mol) in dry tetrahydrofuran (30 ml) and pyridine (4 ml). Stirring was continued overnight and the solution was evaporated to near dryness in vacuum. Water (20 ml) and a few drops of concentrated HCl were added to the residue to acidify it. The precipitate was filtered and washed with saturated solution of sodium bicarbonate (50 ml) and twice with distilled water (50 ml) to give N-substituted succinamic esters (6a, b).
N-substituted succinamic acids (6c, d). To a stirring solution of compound 4 (7.6 mmol) in dry acetone (20 ml), succinic anhydride (0.8 g, 8 mmol) was added and refluxed for 2 h. Acetone was removed with Rota-evaporator. The residue was basified with saturated solution of sodium bicarbonate (10 ml), stirred for 5 min and filtered. The filtrate was acidified with concentrated HCl to pH=2. The precipitate was filtered and then washed twice with distilled water (50 ml). The precipitate was dried to give N-substituted succinamic acids (6c, d).

screening with the potato disc assay
Fresh, disease-free potatoes were obtained from a local market. Tubers of moderate size were surface sterilized by immersion in sodium hypochlorite 0.1% for 20 min. Ends were removed and the potatoes were soaked for an additional 10 min in sodium hypochlorite solution. A core of the tissue was extracted from each tuber with a surfacesterilized 1.0 cm cork borer. Pieces of 2 cm were removed from each end and discarded. The remainder of the cylinder was cut into 0.5 cm discs with a surface-sterilized scalpel. The discs were then transferred to agar plates (1.5 g of agar dissolved in 100 mL double distilled water (DDW), autoclaved for 20 min at 121°C, 20 mL poured into each Petri dish). Each plate contained 5 discs and 3-5 plates, were used for each sample dilution.
A. tumefaciens (ATCC 23341) was cultivated in Soybean Casein Digest Agar. For inoculation of the potato discs, 48 h broth culture containing 5 × 10 9 cells/ml was used. Samples were dissolved in 5% DMSO, filter sterilized, diluted and mixed with the bacterial culture for inoculation.
The potato discs were incubated for 20 days at 25°C incubator, after which Lugol's solution (I 2 /KI) was added, the tumor counts were made and compared with negative controls (bacterial suspension containing 5% DMSO). The results were expressed as + or -percentage versus the number of tumors on the control discs. Significant activity was indicated by consistent negative values of ca. 20% or greater inhibition. Vincristine was used as positive control.
The synthesized compounds appeared as white crystalline substances stable at room temperature. The compounds were insoluble in water and soluble in DMSO.
The proposed structure of compounds was confirmed by IR and 1 H NMR spectroscopy. Interpretation of the 1 H NMR spectra was base on the chemical shifts, multiplicities, and integral intensities of the signals. The yields and physicochemical properties of compounds 3-7 are presented in Table 1.

biological Assay
The antitumor activity of compounds was studied using the potato disc bioassay technique. Minimum inhibitory concentration (MIC) of samples on A. tumefaciens was found to be greater than 1 mg/ml. The formation of formazan pink color developed after addition of MTT dye did not give rise to color at lower concentrations of the tested compounds indicating no inhibition for A. tumefa-ciens growth. The concentrations above the MIC would produce false positive results in potato disc assay due to the antibacterial activity of the compounds and preventing the bacterium to induce the tumogenesis. Therefore, dilutions below MIC including 0.01, 0.1 and 1 mg/ml were used for potato disc assay. The results have been shown in Table 2. Among these N-Benzyl-N'-(4-benzylsulfamoyl-phenyl)-succinamide (7c) and N-Benzyl-N'-[4-(morpholin-4-ylsulfonyl)-phenyl]-succinamide (7d) exhibited significant inhibition of crown gall tumors caused by Agrobacterium tumefaciens at 10 microgram/ml concentration. The activity of compounds 6a, 7b and 7d was comparable to that of vincristine. The results have been shown in Table 2. Vincristine at 0.25 mg/ml was employed